Electron microscopy offers the opportunity to study material architectures in 3D at high resolution. In the field of biological sciences, for example, electron microscopy allows the observation of molecular mechanisms of diseases, the conformation of flexible protein structures and the behavior of individual viruses and proteins in their natural biological context. One technique employed with electron microscopy for analyzing biological materials, for example, is called Slice-and-View™ (hereafter “slice and view”).This technique is typically performed with a dual beam system, that is, a system combining a focused ion beam (FIB) and a scanning electron microscope (SEM) such as the DualBeam® (hereafter “dual beam”) instruments commercially available from FEI Company, the assignee of the present invention.
In the slice and view technique, the FIB cuts and slices a sample with high precision to reveal its 3D internal structures or features. Typically, the FIB exposes a cross section, or face, perpendicular to the top of the surface of the sample material having the hidden feature to be viewed. Because the SEM beam axis is typically at an acute angle relative to the FIB beam axis, a portion of the sample in front of the face is preferably removed so that the SEM beam can have access to image the face. After obtaining an image of the face by the SEM, another layer of substrate at the face may be removed using the FIB, revealing a new, deeper face and thus a deeper cross-section of the feature. Since only the portion of the feature at the very surface of the face is visible to the SEM, sequential repetition of cutting and imaging, or slicing and viewing, provides the data needed to reconstruct the sliced sample into a 3D representation of the feature. The 3D structure is then used to analyze the feature.
The processing of a sample through a slice and view procedure can take a long time if a large section of the sample is processed. This is also true even if the feature of interest is relatively small in relation to the sample because the location of the feature is not typically known with sufficient accuracy to direct the beams of the FIB and SEM to the immediate region of the sample containing the feature. Therefore, a large section of the sample suspected of having the feature is processed in order to locate the feature. With a typical maximum field of view for the SEM being about 150 microns, slice milling and imaging an area this size can be a significant time investment, especially with high resolution settings on the SEM. Alternatively, many smaller portions of the area may be imaged, but doing so generates a vast amount of image data, and the resulting images are typically required to be stitched together to form a larger composite image. Such processes currently can last anywhere from a few hours to several days.
In prior art methods a relatively large section has been required to be processed with every iteration of the slice and view procedure because the shape or direction of the feature through the sample has not been accurately predicted. This problem is especially exacerbated with certain features that have long, winding shapes through the sample, such as is the case with blood vessels or nerves.
In the interest of time, it would be more efficient to slice mill a relatively smaller amount of substrate material necessary to view the feature of interest. Further, it would be more efficient to image a relatively smaller portion of the substrate that contains the feature.